Two-dimensional (2D) cell cultures, in which cells are grown as monolayers, are routinely used as in vitro models of normal and/or diseased tissues for evaluating the effectiveness and/or safety of libraries of molecules with potential as therapeutic drugs. While this screening step precedes preclinical animal studies which are typically required before human clinical trials, cell culture assays can be determinative for initial, yet crucial, “stop/go” decisions on the development of a drug. However, the increasingly recognized role of the in vivo microenvironment in cell response to therapeutic agents and the structural complexity of the living stroma, including cellular barriers, necessitate the incorporation of additional histological and cellular components in in vitro disease models and screening assays.
Three-dimensional (3D) tissue cultures are in vitro models that mimic the architecture of normal and/or diseased tissues. While possible to co-culture with other cells and cellular components that naturally occur in their microenvironment, 3D tissue cultures have been more analogous to conventional monolayer cell cultures than to the in vivo microenvironment of the tissue being modeled.
Furthermore, the usefulness of 3D tissue cultures as in vitro models of normal and/or diseased physiological systems for high throughput screening (HTS) applications and other large-scale studies has been severely limited by technical limitations. For example, existing 3D tissue culture systems do not provide HTS-amenable perfusion of the basolateral space of a cellularized 3D matrix. Specifically, a cellularized 3D matrix in a 3D tissue culture can be coated with a layer of epithelial and/or endothelial cells developed to form a cellular barrier between the cellularized 3D matrix and the surrounding air or cell culture medium. This cellular barrier can reduce or prevent penetration of essential nutrients into the cellularized 3D matrix of the 3D tissue culture and/or exit of toxic metabolic byproducts and waste out of the cellularized 3D matrix of the 3D tissue culture. As a result, the viability of the cells growing in the cellularized 3D matrix is reduced, thus limiting the useful duration time of the 3D tissue culture. One limited solution to this perfusion problem is the use of a transwell system.
FIGS. 1A and 1B illustrate a conventional transwell system supporting a 3D tissue culture. The transwell system includes a transwell vessel 100 with an upper chamber 102 and a lower chamber 104, defined and separated by a semi-permeable membrane 106 that allows perfusion between the chambers. In FIG. 1A, a cellularized 3D matrix 108 is cast in the upper chamber 102 of the transwell and perfused with cell culture medium from the lower chamber 104 via the semi-permeable membrane 106 to begin a 3D tissue culture. A layer of, for example, epithelial or endothelial cells coats the cellularized 3D matrix 108 to form a cellular barrier/interface 110 between the cellularized 3D matrix 108 and air or other media in the upper chamber 102.
In FIG. 1B, the cellularized 3D matrix 108 has contracted in the upper chamber 102 of the transwell vessel 100 to form a contracted 3d matrix 112 that maintains the cellular barrier/interface 110. The majority of 3D tissue cultures involve the use of contractile matrices (e.g., collagen-based gels) and/or matrix-contracting cells (e.g., fibroblasts) that can lead to contraction of a cellularized 3D matrix. Matrix contraction is also a physiological requirement for the in vitro differentiation of some tissue models (e.g., skin). Importantly, this contraction of cellularized 3D matrices during the course of a 3D tissue culture is not only common but also can result in partial or complete detachment of a cellularized 3D matrix from the semi-permeable membrane between the upper chamber and the lower chamber of a transwell vessel, thereby preventing cellular barrier-mediated phase separation (e.g., an air-liquid or liquid-liquid interface).
FIG. 1B shows that detachment of the cellularized 3D matrix 108 (contracted as a result of cell-induced matrix contraction) from the semi-permeable membrane 106 allows the medium in the lower chamber 104 to flood the upper chamber 102. This, in turn, leads to mixture of the two media and loss of a liquid-liquid interface (simulating an endothelial barrier or gastrointestinal epithelium) or loss of an air-liquid interface (simulating skin or lung epithelium). Regardless, the architectural integrity of the transwell system is compromised. Careful macroscopic and/or microscopic examination of each 3D tissue culture in a transwell system is necessary to establish that the functional architecture of any cellularized 3D matrices is intact and that the upper chamber is not flooded with medium from the lower chamber. This makes the use of a transwell system impractical for most HTS applications.
Scalability and compatibility with HTS instruments are also limitations for the use of existing 3D tissue cultures in drug discovery. Industrial-scale HTS efforts probe the biological activity of large chemical libraries consisting of hundreds of thousands of molecules (synthetic or natural extracts) and reagents (e.g., antibodies, peptides, shRNA/siRNA constructs, etc.). Screening protocols make use of industry-standard multi-well plates (including 12, 24, 96, 384, or 1536 wells) for testing molecules and compatible instruments for assay readouts. The generation of cellularized 3D structures in cell culture vessels often requires time-periods of several days, by when cell-extracellular matrix interactions often have already resulted in partial or complete matrix detachment of the structures from the vessel walls (e.g., floating collagen gels).
In parallel, long-term culture conditions necessitate several cycles of vacuum-aspiration of the depleted culture medium and replacement with fresh medium. In conventional 2D cultures, cells are attached to the bottom of the well and therefore automated aspiration of medium from multi-well plates (using multi-channel vacuuming devices) without disrupting the cells is typically feasible. In contrast, 3D tissue cultures are often miniaturized modules of cellularized matrix floating freely within a cell culture vessel. Therefore, aspiration of the medium from a 3D tissue culture vessel has a high probability of causing cellularized 3D matrix loss by aspiration, cellularized 3D matrix damage/fractionation, and/or blockage of a vacuuming channel. These artifactual events can induce major interference with the output of an assay, thus rendering this technical hurdle a major impasse for most efforts to automate HTS efforts with 3D tissue cultures.
Additionally, assay readouts of 3D tissue culture use macroscopic or microscope examination to analyze the structure and/or size of cellularized 3D structures formed within the culture. However, the random positions of floating cellularized 3D structures make 3D tissue cultures poorly suited for automated high-content imaging techniques. Furthermore, automated transfer of cellularized 3D matrices for study is challenging to do without damage/fractionation because the matrices are typically soft and brittle structures.
Accordingly, a need exists for methods, systems, and/or devices for the production, manipulation, and/or analysis of 3D tissue cultures, particularly for HTS applications.